Biology subjects

Liu, F.

Publications and source records attributed to Liu, F..

At least 307 records · Page 17Linked to original sources

bioRxiv2020

Comprehensive genome sequencing analysis as a promising option in the prenatal diagnosis of fetal structural anomalies: a prospective study.

PurposeGenome sequencing (GS) is a powerful tool for postnatal genetic diagnosis, but relevant clinical studies in the field of prenatal diagnosis are few. We aimed to evaluate the feasibility of GS as a first-line approach in prenatal diagnosis and compare its clinical value with the chromosomal microarray analysis (CMA) plus exome sequencing (ES) sequential testing. MethodsWe applied trio GS ([~]40-fold) in parallel with CMA plus ES to investigate the genetic basis for structural or growth anomalies in 111 fetuses and compared their results. ResultsGS covered all genetic variants in 22 diagnosed cases detected by CMA plus ES, yielding a diagnostic rate of 19.8% (22/110). Moreover, GS provided more comprehensive and precise genetic information than CMA plus ES, revealing twin fetuses with an imbalanced translocation arising from a balanced paternal translocation and one fetus with an extra pathogenic variant in the GJA8 gene, and incidentally identified intrauterine CMV infection in a growth-restricted fetus. ConclusionCompared with CMA plus ES, GS offers a more comprehensive view of the genetic etiology of fetal anomalies and provides clues for nongenetic factors such as intrauterine infection. Our study demonstrates the feasibility of GS as a promising first-line test in prenatal diagnosis.

genomics↗

bioRxiv2020

Structure of the activated Roq1 resistosome directly recognizing the pathogen effector XopQ

Plants and animals detect pathogen infection via intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) that directly or indirectly recognize pathogen effectors and activate an immune response. How effector sensing triggers NLR activation remains poorly understood. Here we describe the 3.8 [A] resolution cryo-electron microscopy structure of the activated Roq1, an NLR native to Nicotiana benthamiana with a Toll-like interleukin-1 receptor (TIR) domain, bound to the Xanthomonas effector XopQ. Roq1 directly binds to both the predicted active site and surface residues of XopQ while forming a tetrameric resistosome that brings together the TIR domains for downstream immune signaling. Our results suggest a mechanism for the direct recognition of effectors by NLRs leading to the oligomerization-dependent activation of a plant resistosome and signaling by the TIR domain. One Sentence SummaryVisualization of an activated plant immune receptor that triggers the immune response upon pathogen recognition.

plant biology↗

bioRxiv2020

Rapid host response to an infection with Coronavirus. Study of transcriptional responses with Porcine Epidemic Diarrhea Virus

The transcriptional response in Vero cells (ATCC(R) CCL-81) infected with the coronavirus Porcine Epidemic Diarrhea Virus (PEDV) was measured by RNAseq analysis 4 and 6 hours after infection. Differential expressed genes (DEGs) in PEDV infected cells were compared to DEGs responding in Vero cells infected with Mammalian Orthoreovirus (MRV). Functional analysis of MRV and PEDV DEGs showed that MRV increased the expression level of several cytokines and chemokines (e.g. IL6, CXCL10, IL1A, CXCL8 [alias IL8]) and antiviral genes (e.g. IFI44, IFIT1, MX1, OASL), whereas for PEDV no enhanced expression was observed for these "hallmark" antiviral and immune effector genes. Pathway and Gene Ontology "enrichment analysis" revealed that PEDV infection did not stimulate expression of genes able to activate an acquired immune response, whereas MRV did so within 6h. Instead, PEDV down-regulated the expression of a set of zinc finger proteins with putative antiviral activity and enhanced the expression of the transmembrane serine protease gene TMPRSS13 (alias MSPL) to support its own infection by virus-cell membrane fusion (Shi et al, 2017, Viruses, 9(5):114). PEDV also down-regulated expression of Ectodysplasin A, a cytokine of the TNF-family able to activate the canonical NFKB-pathway responsible for transcription of inflammatory genes like IL1B, TNF, CXCL8 and PTGS2. The only 2 cytokine genes found up-regulated by PEDV were Cardiotrophin-1, an IL6-type cytokine with pleiotropic functions on different tissues and types of cells, and Endothelin 2, a neuroactive peptide with vasoconstrictive properties. Furthermore, by comprehensive datamining in biological and chemical databases and consulting related literature we identified sets of PEDV-response genes with potential to influence i) the metabolism of biogenic amines (e.g. histamine), ii) the formation of cilia and "synaptic clefts" between cells, iii) epithelial mucus production, iv) platelets activation, and v) physiological processes in the body regulated by androgenic hormones (like blood pressure, salt/water balance and energy homeostasis). The information in this study describing a "very early" response of epithelial cells to an infection with a coronavirus may provide pharmacologists, immunological and medical specialists additional insights in the underlying mechanisms of coronavirus associated severe clinical symptoms including those induced by SARS-CoV-2. This may help them to fine-tune therapeutic treatments and apply specific approved drugs to treat COVID-19 patients.

microbiology↗

bioRxiv2020

Cross-regulation between RpfG and HtsHs to regulate antibiotic biosynthesis in Lysobacter

Bacterial two-component systems (TCSs) sense and respond to environmental changes and modulate downstream gene expression. However, the mechanism of cross-talk between multiple TCSs is unclear. In this study, we report a previously uncharacterized mechanism by which the TCS protein RpfG interacts with hybrid two-component system (HyTCS) proteins HtsH1, HtsH2 and HtsH3 to regulate antibiotic biosynthesis in Lysobacter. RpfG, a phosphodiesterase (PDE), can degrade c-di-GMP to 5-pGpG and can regulate antibiotic heat-stable antifungal factor (HSAF) biosynthesis in a PDE- independent manner. Thus, we wondered whether RpfG regulate HSAF biosynthesis through interactions with other factors. Subsequently, we demonstrated that RpfG interacts with three HyTCS proteins (HtsH1, HtsH2 and HtsH3), that can inhibit the PDE enzymatic activity of RpfG. Importantly, deletion of htsH1, htsH2 and htsH3 resulted in significantly decreased HSAF production, and we showed that HtsH1, HtsH2 and HtsH3 depend on their phosphorylation activity to directly regulate HSAF biosynthesis gene expression. Our results reveal that RpfG does not depend on PDE activity to regulate HSAF biosynthesis, rather it interacts with HtsH1, HtsH2 and HtsH3 to do so, a regulatory mechanism that may be a conserved paradigm in Lysobacter and Xanthomonas.

microbiology↗

bioRxiv2020

Neuroinvasive potential of SARS-CoV-2 revealed in a human brain organoid model

Although COVID-19 is considered to be primarily a respiratory disease, SARS-CoV-2 affects multiple organ systems including the central nervous system (CNS). Yet, there is no consensus whether the virus can infect the brain, or what the consequences of CNS infection are. Here, we used three independent approaches to probe the capacity of SARS-CoV-2 to infect the brain. First, using human brain organoids, we observed clear evidence of infection with accompanying metabolic changes in the infected and neighboring neurons. However, no evidence for the type I interferon responses was detected. We demonstrate that neuronal infection can be prevented either by blocking ACE2 with antibodies or by administering cerebrospinal fluid from a COVID-19 patient. Second, using mice overexpressing human ACE2, we demonstrate in vivo that SARS-CoV-2 neuroinvasion, but not respiratory infection, is associated with mortality. Finally, in brain autopsy from patients who died of COVID-19, we detect SARS-CoV-2 in the cortical neurons, and note pathologic features associated with infection with minimal immune cell infiltrates. These results provide evidence for the neuroinvasive capacity of SARS-CoV2, and an unexpected consequence of direct infection of neurons by SARS-CoV-2.

microbiology↗

bioRxiv2020

Performance characterization of PCR-free whole genome sequencing for clinical diagnosis

PurposeTo evaluate the performance of PCR-free whole genome sequencing (WGS) for clinical diagnosis, and thereby revealing how experimental parameters affect variant detection. MethodsAll the 5 NA12878 samples were sequenced using MGISEQ-2000. NA12878 samples underwent WGS with differing DNA input and library preparation protocol (PCR-based versus PCR-free protocols for library preparation). The DP (depth of coverage) and GQ (genotype quality) of each sample were compared. We developed a systematic WGS pipeline for the analysis of down-sampling samples of the 5 NA12878 samples. The performance of each sample was measured for sensitivity, coverage of depth and breadth of coverage of disease-related genes and CNVs. ResultsIn general, NA12878-2 (PCR-free WGS) showed better DP and GQ distribution than NA12878-1 (PCR-based WGS). With a mean depth of ~40X, the sensitivity of homozyous and heterozygous SNPs of NA12878-2 showed higher sensitivity (>99.77% and > 99.82%) than NA12878-1, and positive predictive value (PPV) exceeded 99.98% and 99.07%. The sensitivity and PPV of homozygous and heterozygous indels for NA12878-2 (PCR-free WGS) showed great improvement than NA128878-1. The breadths of coverage for disease-related genes and CNVs are slightly better for samples with PCR-free library preparation protocol than the sample with PCR-based library preparation protocol. DNA input also influences the performance of variant detection in samples with PCR-free WGS. ConclusionDifferent experimental parameters may affect variant detection for clinical WGS. Clinical scientists should know the range of sensitivity of variants for different methods of WGS, which would be useful when interpreting and delivering clinical reports.

genetics↗

bioRxiv2020

A Study of Coronary Artery Disease in the Left Circumflex Artery via Artificial Vessel Restoration

Diagnosis, risk analysis, and treatment of coronary artery disease (CAD) can be improved with a better understanding of cardiovascular flows. Numerical simulations can assist in achieving this understanding. The objective of this study is to compare the dynamics of blood flow in a diseased left circumflex artery (LCX) and its artificially restored counterpart representing its healthy state. This comparison is made to identify flow characteristics in the restored vessel that contribute to the development of CAD. The diseased LCX geometry was derived from computed tomography angiography data. The stenosed region of the diseased LCX was repaired by manually redefining cross-sections of the vessel, creating the restored geometry. To account for inaccuracies, variations of the restored LCX were made by dilating the repaired surface. Numerical simulations were conducted on all geometries and the results were compared. Alongside expected low wall shear stress, a region of high vorticity was present in all of the restored vessels near the location where CAD developed in the diseased vessel. Therefore, this research suggests that flow vorticity is relevant in assessing the risk for CAD, potentially improving the accuracy of non-invasive, computational diagnosis. Such improvements can also help avoid unnecessary invasive diagnosis methods and minimize risk.

bioengineering↗

bioRxiv2020

Mouse model of SARS-CoV-2 reveals inflammatory role of type I interferon signaling

Severe Acute Respiratory Syndrome-Coronavirus 2 (SARS-Cov-2) has caused over 5,000,000 cases of Coronavirus disease (COVID-19) with significant fatality rate.1-3 Due to the urgency of this global pandemic, numerous therapeutic and vaccine trials have begun without customary safety and efficacy studies.4 Laboratory mice have been the stalwart of these types of studies; however, they do not support infection by SARS-CoV-2 due to the inability of its spike (S) protein to engage the mouse ortholog of its human entry receptor angiotensin-converting enzyme 2 (hACE2). While hACE2 transgenic mice support infection and pathogenesis,5 these mice are currently limited in availability and are restricted to a single genetic background. Here we report the development of a mouse model of SARS-CoV-2 based on adeno associated virus (AAV)-mediated expression of hACE2. These mice support viral replication and antibody production and exhibit pathologic findings found in COVID-19 patients as well as non-human primate models. Moreover, we show that type I interferons are unable to control SARS-CoV2 replication and drive pathologic responses. Thus, the hACE2-AAV mouse model enables rapid deployment for in-depth analysis following robust SARS-CoV-2 infection with authentic patient-derived virus in mice of diverse genetic backgrounds. This represents a much-needed platform for rapidly testing prophylactic and therapeutic strategies to combat COVID-19.

immunology↗

bioRxiv2020

Osteoarthritis: articular chondrocyte inflammatory signaling leads to enhanced gene expression and function of mechanotransduction channel Piezo1 as a pathogenic feedforwardmechanism

Osteoarthritis (OA) is a painful and debilitating condition of synovial joints without any disease-modifying therapies (1, 2). We previously identified mechanosensitive PIEZO channels, PIEZO1 and PIEZO2, both expressed in articular cartilage, to function in chondrocyte mechanotransduction in response to injury (3, 4). We therefore asked whether interleukin-1-mediated inflammatory signaling, as occurs in OA, influences Piezo gene expression and channel function, thus indicative of maladaptive reprogramming that can be rationally targeted. Primary porcine chondrocyte culture and human osteoarthritic cartilage tissue were studied. We found that interleukin-1 (IL-1) upregulated Piezo1 in porcine chondrocytes. Piezo1 expression was significantly increased in human osteoarthritic cartilage. Increased Piezo1 expression in chondrocytes resulted in a feed-forward pathomechanism whereby increased function of Piezo1 induced excess intracellular Ca2+, at baseline and in response to mechanical deformation. Elevated resting state Ca2+ in turn rarefied the F-actin cytoskeleton and amplified mechanically-induced deformation-microtrauma. As intracellular substrates of this novel OA-related inflammatory pathomechanism, in porcine articular chondrocytes exposed to IL-1 we discovered that enhanced Piezo1 expression depended on p38 MAP-kinase and transcription factors HNF4 and ATF2/CREBP1. CREBP1 directly bound to the proximal PIEZO1 gene promoter. In ensemble, these signaling and genetic re-programming events represent a novel and detrimental Ca2+-driven feed-forward mechanism that can be rationally targeted to stem the progression of OA. Significance StatementOsteoarthritis affecting weight-bearing joints is a global health problem, causing loss of mobility and enormous healthcare costs. Disease-modifying approaches are lacking. Here, we report a new cellular mechanism of inflammatory signaling in chondrocytes, the cellular substrate of cartilage. We show how osteoarthritis-relevant levels of interleukin-1 reprogram articular chondrocytes so that they become more susceptible to mechanical trauma, which chondrocytes sense via Piezo1/2 mechanosensitive ion channels. We uncover that IL-1 enhances gene expression of Piezo1 in primary articular chondrocytes underlying Piezo1 gain-of-function. We elucidate the new signaling pathway, from membrane to nucleus, including transcription factors that enhance Piezo1-expression. We also define detrimental effects of gain-of-function of Piezo1, for mechanotransduction and at-rest, that suggest this new reprogramming mechanism to contribute to osteoarthritis pathogenesis.

molecular biology↗

bioRxiv2020

Gene Editing Reveals Obligate and Modulatory Components of the CO2 Receptor Complex in the Malaria Vector Mosquito, Anopheles coluzzii

The sensitivity to volatile carbon dioxide (CO2) produced by humans and other animals is a critical component in the host preference behaviors of the malaria vector mosquito Anopheles coluzzii. The molecular receptors responsible for the ability to sense CO2 are encoded by three putative gustatory receptor (Gr) genes (Gr22,23,24) which are expressed in a distinctive array of sensory neurons housed in maxillary palp capitate peg sensilla of An. coluzzii. Despite the identification of these components and subsequent studies, there is a paucity of understanding regarding the respective roles of these three GRs in the mosquitos CO2 transduction process. To address this, we have used CRISPR/Cas9-based gene editing techniques combined with in vivo electrophysiological recordings to directly examine the role of Gr22,23,24 in detecting CO2 in An. coluzzii. These studies reveal that both Gr23 and Gr24 are absolutely required to maintain in vivo CO2 sensitivity while, in contrast, Gr22 knock out mutants are still able to respond to CO2 stimuli albeit with significantly weaker sensitivity. Our data supports a model in which Gr22 plays a modulatory role to enhance the functionality of Gr23/24 complexes that are responsible for CO2 sensitivity of mosquitoes.

neuroscience↗

bioRxiv2020

Expanding the depth and sensitivity of cross-link identification by differential ion mobility using FAIMS

In cross-linking mass spectrometry, the depth and sensitivity is often limited by the low abundance of cross-links compared to non-cross-linked peptides in the digestion mixture. To improve the identification efficiency of low abundant cross-links, here we present a gas-phase separation strategy using high field asymmetric waveform ion mobility spectrometry (FAIMS) coupled to the Orbitrap Tribrid mass spectrometers. By enabling an additional peptide separation step in gas phase using the FAIMS device, we increase the number of cross-link identification by 23% for a medium complex sample and 56% for strong cation exchange-fractionated HEK293 cell lysate. Furthermore, we show that for medium complex samples, FAIMS enables the collection of single-shot cross-linking data with comparable depth to the corresponding sample fractionated by chromatography-based approaches. Altogether, we demonstrate FAIMS is highly beneficial for XL-MS studies by expanding the proteome coverage of cross-links while improving the efficiency and confidence of cross-link identification.

biochemistry↗

bioRxiv2020

Double-targeted knockdown of miR-21 and CXCR4 inhibits malignant glioma progression by suppression of the PI3K/AKT and Raf/MEK/ERK pathways

BackgroundCurrently, miR-21 and CXCR4 are being extensively investigated as two unrelated key regulators in glioma malignancy. In this study, we investigated the combined effect of these two factors on glioma progression. MethodsWe confirmed the expression of miR-21 and CXCR4 in malignant glioma tissue and glioma cells with qRT-PCR and western blotting. Single-targeted knockdown of miR-21 and CXCR4, as well as double-targeted knockdown of miR-21 and CXCR4 lentiviral vectors were constructed and they were transfected to U87 and U251 cells. Cell proliferation, apoptosis, invasion, and migration from different treatment groups were assessed by MTT assay, Flow Cytometry analysis, Transwell analysis, and Scratch assay, respectively. U87 xenograft mice were constructed to detect roles and potential mechanisms of miR-21 and CXCR4 in malignant glioma tumor growth. ResultsThe expression of miR-21 and CXCR4 was increased in tumor tissues and cell lines. Inhibition of miR-21, CXCR4, and miR-21 and CXCR4 together all reduced the migration, invasiveness, proliferation and enhanced apoptosis in glioma cells, as well as reduced tumor volume and mass in xenograft model. The inhibition effect was strongest in double-targeted knockdown of miR-21 and CXCR4 group, whose downstream pathways involved in AKT axis and ERK axis activation. ConclusionsOur findings reported that double-targeted knockdown of miR-21 and CXCR4 could more effectively inhibit the proliferation, migration, invasion and growth of transplanted tumor and promote cell apoptosis, which were involved in the PI3K/AKT and Raf/MEK/ERK signaling pathways.

cancer biology↗

bioRxiv2020

Insulin/IGF signaling and TOR promote vitellogenesis via inducing juvenile hormone biosynthesis

Vitellogenesis, including vitellogenin (Vg) production in the fat body and Vg uptake by maturing oocytes, is of great importance for the successful reproduction of adult females. The endocrinal and nutritional regulation of vitellogenesis differs distinctly in insects. Here, the complex crosstalk between juvenile hormone (JH) and the two nutrient sensors, insulin/IGF signaling (IIS) and target of rapamycin (TOR), was investigated to elucidate the molecular mechanisms regulating vitellogenesis in the American cockroach, Periplaneta americana. Our data showed that a block of JH biosynthesis or JH action arrested vitellogenesis, partially by inhibiting the expression of doublesex (Dsx), a key transcription factor gene involved in the sex determination cascade. Depletion of IIS and TOR blocked both JH biosynthesis and vitellogenesis. Importantly, the JH analog methoprene, but not bovine insulin (to restore IIS) and amino acids (to restore TOR activity), restored vitellogenesis in the neck-ligated (both nutrient- and JH-deficient) cockroaches. Combining classic physiology with modern molecular techniques, we have demonstrated that JH signaling alone is able to induce vitellogenesis and thus ovarian maturation. By contrast, IIS and TOR do not induce vitellogenesis independent of JH, the nutrient sensors promote vitellogenesis in an indirect manner via activating JH biosynthesis.

developmental biology↗

bioRxiv2020

Characterizing sensitivity and coverage of clinical WGS as a diagnostic test for genetic disorders

BackgroundWith the reduce of cost and incomparable advantages, WGS is likely to change the way of clinical diagnosis of rare and undiagnosed diseases. However, the sensitivity and breadth of coverage of clinical WGS as a diagnostic test for genetic disorders has not been fully evaluated, especially for CNV detection. MethodsAll the samples underwent WGS using MGISEQ-2000. The performance of NA12878, YH cell line, and the Chinese trios were measured for sensitivity, PPV, depth and breadth of coverage. We also compared the performance of WES and WGS using NA12878. The sensitivity and PPV were tested using family-based trio design in the Chinese trios. We also developed a systematic WGS pipeline for the analysis of 8 clinical cases with known disease-causing variants. ResultsIn general, the sensitivity and PPV for SNV/indel detection increased with mean depth, and reached a plateau at a ~40X mean depth using down-sampling samples of NA12878. With a mean depth of 40X, the sensitivity of homozyous and heterozygous SNPs of NA12878 was >99.25% and >99.50% respectively, and PPVs were 99.97% and 98.96%. Homozygous and heterozygous indels showed lower sensitivity and PPV. The sensitivity and PPV is still not 100% even with a mean depth of ~150X. We also observed a substantial variation in the sensitivity of CNV detection across different tools, especially in CNVs with a size of less than 1kb. In general, the breadth of coverage for disease-associated genes and CNVs increased with mean depth. The sensitivity and coverage of WGS (~40X) is better than WES (~120X). Among the Chinese trios with ~40X mean depth, the sensitivity in the offspring was >99.48% and >96.36% for SNP and indel detection, and PPVs were 99.86% and 97.93%. All the 12 previously validated variants in the 8 clinical cases were successfully detected by our WGS pipeline. ConclusionsThe current standard of a mean depth of 40X may be sufficient for SNV/indel detection and identification of most CNVs. Clinical scientists should know the range of sensitivity and PPV for different classes of variants for a particular WGS pipeline, which would be useful when interpreting and delivering clinical reports.

genomics↗

bioRxiv2020

The ModelSEED Database for the integration of metabolic annotations and the reconstruction, comparison, and analysis of metabolic models for plants, fungi, and microbes

For over ten years, ModelSEED has been a primary resource for the construction of draft genome-scale metabolic models based on annotated microbial or plant genomes. Now being released, the biochemistry database serves as the foundation of biochemical data underlying ModelSEED and KBase. The biochemistry database embodies several properties that, taken together, distinguish it from other published biochemistry resources by: (i) including compartmentalization, transport reactions, charged molecules and proton balancing on reactions;; (ii) being extensible by the user community, with all data stored in GitHub; and (iii) design as a biochemical "Rosetta Stone" to facilitate comparison and integration of annotations from many different tools and databases. The database was constructed by combining chemical data from many resources, applying standard transformations, identifying redundancies, and computing thermodynamic properties. The ModelSEED biochemistry is continually tested using flux balance analysis to ensure the biochemical network is modeling-ready and capable of simulating diverse phenotypes. Ontologies can be designed to aid in comparing and reconciling metabolic reconstructions that differ in how they represent various metabolic pathways. ModelSEED now includes 33,978 compounds and 36,645 reactions, available as a set of extensible files on GitHub, and available to search at https://modelseed.org and KBase.

systems biology↗

bioRxiv2020

Structure-Based Design, Synthesis and Biological Evaluation of Peptidomimetic Aldehydes as a Novel Series of Antiviral Drug Candidates Targeting the SARS-CoV-2 Main Protease

SARS-CoV-2 is the etiological agent responsible for the COVID-19 outbreak in Wuhan. Specific antiviral drug are urgently needed to treat COVID-19 infections. The main protease (Mpro) of SARS-CoV-2 is a key CoV enzyme that plays a pivotal role in mediating viral replication and transcription, which makes it an attractive drug target. In an effort to rapidly discover lead compounds targeting Mpro, two compounds (11a and 11b) were designed and synthesized, both of which exhibited excellent inhibitory activity with an IC50 value of 0.05 M and 0.04 M respectively. Significantly, both compounds exhibited potent anti-SARS-CoV-2 infection activity in a cell-based assay with an EC50 value of 0.42 M and 0.33 M, respectively. The X-ray crystal structures of SARS-CoV-2 Mpro in complex with 11a and 11b were determined at 1.5 [A] resolution, respectively. The crystal structures showed that 11a and 11b are covalent inhibitors, the aldehyde groups of which are bound covalently to Cys145 of Mpro. Both compounds showed good PK properties in vivo, and 11a also exhibited low toxicity which is promising drug leads with clinical potential that merits further studies.

biochemistry↗

bioRxiv2020

Structure of RNA-dependent RNA polymerase from 2019-nCoV, a major antiviral drug target

A novel coronavirus (2019-nCoV) outbreak has caused a global pandemic resulting in tens of thousands of infections and thousands of deaths worldwide. The RNA-dependent RNA polymerase (RdRp, also named nsp12), which catalyzes the synthesis of viral RNA, is a key component of coronaviral replication/transcription machinery and appears to be a primary target for the antiviral drug, remdesivir. Here we report the cryo-EM structure of 2019-nCoV full-length nsp12 in complex with cofactors nsp7 and nsp8 at a resolution of 2.9-[A]. Additional to the conserved architecture of the polymerase core of the viral polymerase family and a nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain featured in coronaviral RdRp, nsp12 possesses a newly identified {beta}-hairpin domain at its N-terminal. Key residues for viral replication and transcription are observed. A comparative analysis to show how remdesivir binds to this polymerase is also provided. This structure provides insight into the central component of coronaviral replication/transcription machinery and sheds light on the design of new antiviral therapeutics targeting viral RdRp. One Sentence SummaryStructure of 2019-nCov RNA polymerase.

biochemistry↗

bioRxiv2020

Quantifying temperature compensation of Bicoid gradients with a fast T-tunable microfluidic device

As a reaction-diffusion system strongly affected by temperature, the early fly embryos surprisingly show highly reproducible and accurate developmental patterns during embryogenesis under temperature perturbations. To reveal the underlying temperature compensation mechanism, it is important to overcome the challenge in quantitative imaging on fly embryos under temperature perturbations. Inspired by a microfluidics generating temperature steps on fly embryos, here we design a microfluidic device capable of ensuring the normal development of multiple fly embryos as well as achieving real-time temperature control and fast temperature jumps for quantitative live imaging with a home-built two-photon microscope. We apply this system to quantify the temperature compensation of the morphogen Bicoid (Bcd) gradient in fly embryos. The length constant of the exponential Bcd gradient reaches the maximum at 25 {degrees}C within the measured temperatures of 18-29 {degrees}C and gradually adapts to the corresponding value at new temperatures upon a fast temperature switch. Such an adaption decreases to a less degree if temperature is switched in a later developmental stage. This age-dependent temperature compensation could not be explained with the traditional synthesis-diffusion-degradation (SDD) model assuming the static parameters but an extended SDD model incorporating the dynamic change of the parameters controlling the formation of Bcd gradients. SIGNIFICANCEThermal robustness is important for biological systems experiencing temperature fluctuations. To reveal the temperature compensation mechanism, the fruit fly embryo is an ideal model system. It is intriguing how the early fly embryo achieves highly reproducible and accurate patterning despite it is a reaction-diffusion system strongly affected by temperature. However, it has been challenging to quantitatively measure the developmental patterns in fly embryos under temperature perturbations. To overcome this problem, we construct a fast temperature tunable microfluidic device for fly embryos. Combining quantitative imaging with this device and mathematical modeling, we successfully quantify the temperature response of the morphogen Bicoid (Bcd) gradient and reveal that the temperature compensation for the Bcd gradient is stronger in the later developmental stage.

developmental biology↗